Department of Microbial Biotechnology, Rajiv Gandhi Institute of IT and Biotechnology, Bharati Vidyapeeth Deemed University 411046, Pune, India*Corresponding author: [email protected]
Comparative studies on potential probioticcharacteristics of Lactobacillus acidophilus strains
1
EurAsian Journal of BioSciences Eurasia J Biosci 7, 1-9 (2013)http://dx.doi.org/10.5053/ejobios.2013.7.0.1
Lactobacilli are often considered to be
commensal or beneficial participants in human
microbial ecology and considerable research is being
carried out on the effects for the use of lactobacilli
as additives in both human and animal diets
(Hummel et al. 2007). In recent years, the probiotic
activity of lactic acid bacteria (Lactobacilli,
Streptococci and Bifidobacteria) has been
emphasized. A number of health benefits have been
claimed for probiotic bacteria and are also being
recommended as a preventive approach to maintain
the balance of intestinal microflora (Shah 2007).
Their beneficial effects on humans include i)
stabilization of intestinal microflora (excluding
colonization of entero-pathogenic bacteria by
adhesion to the intestinal wall and competition for
nutrients) (Denev 2006), ii) reduction of lactose
intolerance (de Vrese et al. 2001), iii) prevention of
antibiotic-induced diarrhoea (Pochapin 2000), iv)
prevention of colon cancer (Wollowski et al. 2001), v)
stimulation of the immune system (Isolauri et al.
2001) etc.
Probiotic microorganisms should express high
tolerance to acid and bile and ability to adhere to
intestinal surfaces to survive in and colonise in
gastro-intestinal tract (GIT). However in vivo testing
is expensive, time consuming and requires approval
by ethical committees. Hence, reliable in vitro
methods for selection of promising strains have
been used by earlier researchers (Jacobsen et al.
1999). One of the unique features of probiotics is
their antibiotic resistance expression and
transferability as there is great concern over
possible spread of resistance determinants to
Received: August 2012Accepted: December 2012
Printed: January 2013
INTRODUCTION
AbstractBackground: Probiotics are live microorganisms present in food and dietary supplements thatbeneficially affect the individual by improving the intestinal microbial balance properties. Theirmarket value and biological potential is enormous because of their health-promoting properties.Therefore, comparative studies on probiotic potential of three selected strains of Lactobacillusacidophilus were carried out during this work.Method: The selected strains of Lactobacillus acidophilus (NCIM 2660, NCIM 2903 and NCIM 2285)were assessed for tolerance to pH (pH 2.5) and oxgall (0.3%), adhesion to Caco-2 cells, antimicrobialproperties against test microorganisms and susceptibility to antibiotics. Results: The distinguished characteristics of strain NCIM 2903 were high acid tolerance,antagonistic activity against all test micro-organisms and adhesion to Caco-2 cells (225±33 cellsadhering to 100 Caco-2 cells). Strain NCIM 2285 exhibited bile tolerance and inability to grow inpresence of five of the antibiotics used. Strain NCIM 2285 indicated moderate adhesion to Caco-2cells. The strain NCIM 2660 was unable to grow in presence of any of the twelve antibiotics. Incontrast, control plates without discs of antibiotics exhibited confluent growth. It was a desirablefeature. However, NCIM 2660 exhibited poor adhesive property. The optimum temperature ofgrowth for all three strains was found to be 37ºC. Conclusions: Significant differences in the probiotic characteristics of the three strains wererecorded. In the cumulative assessment, strain NCIM 2903 could be regarded as the potentialprobiotic contender. These studies will help in selecting the probiotic micro organisms in a morerational manner for further applications.Keywords: Acid and bile tolerance, antibiotic susceptibility, antimicrobial activity, cell adhesion,lactic acid bacteria.
Dixit G, Samarth D, Tale V, Bhadekar R (2013) Comparative studies on potential probioticcharacteristics of Lactobacillus acidophilus strains. Eurasia J Biosci 7: 1-9.
http://dx.doi.org/10.5053/ejobios.2013.7.0.1
Gauri Dixit, Deepti Samarth, Vidya Tale, Rama Bhadekar*
©EurAsian Journal of BioSciences
human pathogenic and opportunistic bacteria
(Ammor et al. 2007).
Lactobacilli are highly competitive largely due to
their applications in the production of fermented
food. They can also produce antimicrobial
substances including bacteriocins that have ability to
inhibit pathogenic and food spoilage bacteria
(Rattanachaikunsopon and Phumkhachorn 2010).
These compounds have shown to exert specific
antagonistic properties against Gram-negative and
Gram-positive pathogens. Adhesion of lactic acid
bacteria (LAB) to mucosal surfaces has been studied
in vitro using Caco-2 cells (Duary et al. 2011).
The aforementioned points indicate that
screening and selection of novel probiotic strains is
critical, thereby depending on definite criteria. Most
efficient strains will be the strains that are robust
enough to survive the harsh physico-chemical
conditions present in GIT.
Lactobacillus acidophilus is a well known and well
studied probiotic microorganism. However, it is now
clear that different strains undoubtedly vary in their
efficiency and probiotic potentials (Ng et al. 2009).
Hence the present study is an effort to give a
comparative account of three strains of L.
acidophilus in the group of probiotic bacteria.
All media components were purchased from Hi
Media, India. All chemicals were purchased from
Merck (India) and all were of A.R. grade.
Bacterial strains
Bacterial cultures were procured from National
Collection of Industrial Microorganisms (NCIM),
National Chemical Laboratory, Pune, India. Three
strains of Lactobacillus acidophilus used for
comparative studies were NCIM 2660, NCIM 2285
and NCIM 2903. They were maintained by
subculturing on MRS (De Man, Rogosa and Sharpe)
agar under microaerophillic conditions. Test
microorganisms viz. Pseudomonas aeruginosa (NCIM
2200), Staphylococcus aureus (NCIM 5021),
Escherichia coli (NCIM 2065), Klebsiella pneumoniae
(NCIM 5082) and a clinical isolate of Salmonella Typhi
(Salmonella enterica enterica, serovar Typhi) were
assessed for experiments of antimicrobial activity.
They were maintained on nutrient agar. The stock
cultures were preserved in 10% glycerol and were
subcultured routinely at the interval of every two
months. The cultures were stored at 4°C between
transfers and were subcultured once before
experimental use.
Epithelial cell line culture
The enterocyte-like Caco-2 cell line was obtained
from the National Centre for Cell Sciences (NC CS,
Pune, India) and grown on Eagle’s Minimum
essential medium (MEM) supplemented with 5.0
mmol L-1 L-glutamine, 20 mmol L-1 D-glucose, 1.0
mmol L-1 sodium pyruvate and 20% heat-inactivated
(30 min; 56°C) foetal calf serum (Gibco, Germany).
For the adhesion assays, cells were prepared on a 25
cm2 plastic tissue culture flask. They were used,
between the 40th and 45th passage after 21 days of
incubation at 37°C.
For all experiments 10% inoculi of late log phase
cultures (equivalent to the 0.5 Mcfarland turbidity
standards) were used.
Effect of temperature on the growth of L.
acidophilus strains
Effect of temperature on viability of L. acidophilus
strains was examined by inoculating them in MRS
broth and incubating at 28ºC, 37ºC and 45ºC. After
the incubation period of 24 h, growth was
determined by measuring the optical density at 600
nm.
Determination of acid tolerance
Percent survival of the three strains was
determined after exposure to pH 2.5 for 2 h and 4 h
at 37°C. For this, overnight grown cultures were
inoculated in MRS broth adjusted to pH 2.5. The
samples were plated onto MRS agar at the end of
exposure time. The plates were incubated for 24 h at
37°C and total viable count was determined. Un-
inoculated broth served as negative control while
set of test organisms inoculated in MRS broth (pH
6.2) was used as positive control.
Determination of bile tolerance
Bile tolerance of L. acidophilus cultures was
examined by inoculating them in MRS broth
containing oxgall (Central Drug House, New Delhi,
India) at the concentrations of 0.2%, 0.3% and 0.5%.
Dixit et al.
2
EurAsian Journal of BioSciences 7: 1-9 (2013)
MATERIALS AND METHODS
The control comprised of MRS broth without bile
salt. Bacterial growth was monitored by measuring
absorbance at 600 nm after incubation for 24 h at
37°C.
Antimicrobial activity
Using in-vitro agar well diffusion method,
antimicrobial activity experiments were carried out
(Zinedine and Faid 2007). The activity of
Lactobacillus strains against test microorganisms
mentioned earlier was recorded. Overnight grown
cultures of L. acidophilus strains were inoculated in
the wells of nutrient agar where as test
microorganisms were inoculated by pour plate
technique. The plates were incubated at 37ºC for 24
h. The inhibition zones were measured at the end of
incubation period.
Antibiotic susceptibility assay
A disc diffusion assay was performed to study
antibiotic susceptibility of L. acidophilus strains. The
cultures were inoculated in MRS agar using pour
plate technique. The antibiotics were supplied in the
form of dodeca discs (Hi Media, India) which included
nalidixic acid, norfloxacin, co-trimoxazole,
gentamycin, ampicillin, cephalexin, mecillinam,
chloramphenicol, tetracycline, kanamycin, amikacin
and streptomycin. The zones of inhibition were
measured after incubation at 37ºC for 24 h. The
strains were cultivated on control plates (MRS agar
without antibiotic discs) under identical conditions.
Adhesion assay
The adherence of L. acidophilus strains to Caco-2
cells was examined as described previously by
Jacobsen et al. (1999). The adherent lactobacilli in 25
random microscopic fields were counted for each
test. Bacterial strains were scored as non-adhesive
when fewer than 60 bacteria were present in 25
fields, adhesive with 61 to 100 bacteria in 25 fields,
and strongly adhesive with more than 100 bacteria
in 25 fields.
Statistical analysis
All experiments in the present study were carried
out in triplicates and the results indicate their mean
values. For statistical analysis, the standard errors of
the means were calculated and the means were
tested according to One-way ANOVA test for
significant differences among the samples. A
statistical significance was accepted at significant
level P<0.05.
In this work, in vitro evaluation of certain
properties of L. acidophilus strains important for
their survival in GIT has been carried out.
Effect of temperature on the growth of L.
acidophilus strains
In all the three experiments, effect of tempera-
tures 28ºC, 37ºC and 45ºC on the growth of three
selected strains were found to be significantly
different (P<0.05). Fig. 1 shows the effect of
temperature on viability on L. acidophilus strains. All
strains grew well at 28ºC and 37ºC and their growth
was comparable. None of them could grow at 45ºC.
Considering the physiological temperature of
human body and the results obtained, all further
experiments were carried out at 37ºC.
Determination of acid tolerance
Probiotic bacteria are mostly delivered in a food
system and must be acid and bile tolerant to survive
in the human gastrointestinal tract. The time from
entrance to release from the stomach has been
estimated to be approximately 90 min with further
digestive processes requiring longer residence time
(Berrada et al. 1991). Table 1 shows survival of L.
acidophilus strains after exposure to pH 2.5 for 2 h
and then after 4 h. The survival rates of all three
strains were higher after 2 h incubation period
(P<0.05) than 4 h (P<0.05) at pH 2.5. The percent
survival of strain NCIM 2285 decreased by almost
50% after 4 h as compared to growth after 2 h. In
case of strains NCIM 2903 and NCIM 2660 percent
survival decreased by 21% and 43% respectively. The
results suggested that strain 2903 had better acid
tolerance among the three strains.
Acid tolerance of Lactobacillus strains was also
reported by Jacobsen et al. (1999) who recorded 4
out of 8 strains of L. acidophilus capable of survival at
pH 2.5 for 2 h. Our results indicating the survival of
L. acidophilus (particularly strain NCIM 2903) at pH
2.5 after 4 h were very significant.
Determination of bile tolerance
The ability of Lactobacillus strains to grow in
3
Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)
RESULTS AND DISCUSSION
presence of bile salts was studied by growing them
in MRS broth supplemented with different
concentrations of oxgall (0.2 to 0.5%). Bile tolerance
is an important characteristic of probiotic
microorganisms. A concentration of 0.3% oxgall
closely appropriates the bile level found in the
gastrointestinal tract (Goldin and Gorbach 1992).
Fig. 2 clearly reveals that the strains were viable at
different concentrations of bile salt and showed
maximum growth at 0.2% bile. At 0.3% bile
concentration strain, NCIM 2660 exhibited weak
growth whereas at still higher concentration of
0.5%, both NCIM 2660 and NCIM 2903 showed
negligible growth indicating their inability to
tolerate the high bile concentration. Thus, strain
NCIM 2285 was found to be most bile tolerant
among the three strains. The three strains exhibited
significant difference (P<0.05) in their bile tolerance.
Many reports recorded the bile tolerance of lactic
acid bacteria (LAB). However, majority of them
demonstrated bile tolerance upto 0.3%
concentration (Liong and Shah 2005, Mcauliffe et al.
2005). Therefore our results for strain NCIM 2285
are noteworthy taking into account its high bile
tolerance.
Antimicrobial activity
The antimicrobial properties of L. acidophilus
strains varied with the test microorganisms (Table
2). Strain NCIM 2903 had astonishingly antagonistic
property towards test microorganisms taken under
consideration and the zones of inhibition also varied
from medium to large. The other two strains had
antagonistic activity against only 3 out of 5 test
microorganisms. Strain NCIM 2260 showed weak
zones of inhibition against P. aeruginosa and K.
pneumoniae and could not inhibit S. aureus and E.
coli. Strain NCIM 2285 could not inhibit P. aeruginosa
and Salmonella Typhi.
Our results are in line with reports by Jacobsen et
al. (1999). They reported only 3 amongst 8 strains of
L. acidophilus strains showing weak zone of
inhibition against S. aureus. None of them could
strongly inhibit E. coli. Hence our results of strain
NCIM 2903 showing inhibitory activity against all
test organisms are very promising, thereby
emphasizing its probiotic characteristics. LABS have
been shown to possess inhibitory activity towards
the growth of pathogenic bacteria such as Listeria
monocytogenes (Sifour et al. 2012), E. coli, Salmonella
spp. (Hudault et al. 1997, Makras et al. 2006) and
others (Olsen et al. 1995, Coconnier et al. 1997).
The mechanism(s) of antibacterial activity in
probiotic Lactobacillus strains appears to be
multifactorial (Servin 2004) and is due to
bacteriocins and/or organic acid produced by them.
Inhibition of pathogen cultured from pancreatic
necrosis due to the production of organic acids by
multi-species probiotic was noted by Ridwan et al.
(2008). Lactobacilli have been shown to possess
inhibitory activity towards the multiplication of
enteropathogens (Drago et al. 1997). The
antagonistic activity is variable and more strain-
specific (Jacobsen et al. 1999). Bacteriocins
produced by LAB may also prove useful for different
gastrointestinal and uro-genital infection therapies
and also to overcome problem of antimicrobial
resistance (Šušković et al. 2010).
Antibiotic susceptibility assay
The determination of antibiotic susceptibility of a
bacterial strain is an important prerequisite prior to
considering it safe for human and animal
consumption. All the three strains were subjected to
antibiotic susceptibility test. The control plates
showing confluent growth under identical
conditions were used to compare the results of agar
disc diffusion assay. The results are summarised in
Table 3. Inhibition zones were not observed for the
strains NCIM 2903 and NCIM 2285 in presence of
norfloxacin and mecillinam amongst the 12
antibiotics used while zones of inhibition varying
from 2 mm to 15 mm with rest of the antibiotics
were recorded. Strain NCIM 2660 did not show
growth in presence of any of the antibiotics used.
According to earlier reports, specific antibiotic
4
Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)
Table 1. Effect of pH (2.5) on survival of L. acidophilusstrains.
5
Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)
Fig. 1. Effect of temperature on the growth of L. acidophilus strains.
Fig. 2. Effect of bile concentration on growth of L. acidophilus strains.
Table 2. In vitro antagonistic activity of L. acidophilus strains against test microorganisms.
-- : Absence of zone of inhibition, SD: Standard deviation
resistance traits among probiotic strains may be
desirable (Charteris et al. 1998). Some authors claim
that in cases of co-administration with antibiotics to
prevent and treat intestinal disorders, probiotics
should be resistant to certain antibiotics so as to
survive in the gastrointestinal tract. However, this
opinion is controversial.
Probiotics containing resistance traits may have
negative consequences to human health. The
presence of antibiotic-resistance genes in many LAB,
and the transfer of plasmids and conjugative
transposons (Yoshiyuki et al. 2009) to and from LAB,
have been reported in Lactobacillus species. The
resistance to chloramphenicol, kanamycin,
erythromycin, gentamycin, streptomycin and
tetracycline (also considered in present work) had
shown to be plasmid borne in certain probiotic
cultures (Temmerman et al. 2002, Moubareck et al.
2005, Hummel et al. 2007). Thus, there exists risk
relating to potential transfer of antibiotic resistance
from probiotic strains to other bacteria either
commensally residing in intestine and/or pathogens
is undesirable and detrimental. Studies by Hummel
et al. (2007) demonstrated that resistant genes
might be present in probiotic strains but are silent.
Genetic basis and associated resistance mechanisms
towards some antibiotics are still unknown.
Adhesion assay
The adhesion of 3 different L. acidophilus strains
was studied using Caco-2 cell line as a model for
intestinal epithelium (P<0.05). The adherence of
bacteria was observed microscopically. Table 4
indicates the number of Lactobacilli exhibited in
vitro adherence to Caco-2 cells. All the three strains
exhibited varying degree of adhesion. The most of
the adhesive strain was L. acidophilus NCIM 2903.
Strain NCIM 2285 was moderately adhesive whereas
strain NCIM 2660 was unable to adhere to this
intestinal cellular model. L. acidophilus strains either
unable to adhere to Caco-2 cells or showing weak
adhesion have been reported by Sareml et al. (1996)
and Jacobsen et al. (1999). However one of the
selected strains (NCIM 2903) showing significant
adhesion to Caco-2 cells that would satisfy major
prerequisite as probiotic.
In conclusion, the study reported in vitro methods
that could be exploited for prediction of the survival
potential of Lactobacilli in human GIT since it is
essentially linked to adhesion to Caco-2 cells and
acid and bile tolerance. Thus, the comparison of
potential probiotic traits of examined strains
revealed NCIM 2903 as a promising candidate.
6
Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)
Table 3. Antibiotic resistance of L. acidophilus strains using disc diffusion method.
bMeans in the same row followed by superscript letter are significantly different (P<0.05).NG - no growth -- = inhibition zone not observed.C= Chloramphenicol; A = Ampicillin; T= Tetracycline; G = Gentamycin; K= Kanamycin; Co= Co-trimoxazole; Ak= Amikacin; S= Streptomycin;No= Norfloxacin; Nx= Nalidixic acid; Cp= Cephalexin; Mc= Mecillinam.
Table 4. Adhesion of L. acidophilus strains to Caco-2 cells inculture.
*Expressed as the number of lactobacilli adhering per 100epithelial Caco-2 cells ; mean±standard deviations of three assays,counts were carried out on 25 randomized microscopic fields foreach sample per assay.
7
Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)
Ammor MS, Florez AB, Mayo B (2007) Antibiotic resistance in non-enterococcal lactic acid bacteria and Bifidobacteria.Food Microbiology 24(6): 559-570. http://dx.doi.org/10.1016/j.fm.2006.11.001
Berrada N, Lemeland G, Laroch P, Thouveno TP, Piaia M (1991) Bifidobacterium from Fermented Milks: Survival DuringGastric Transit. Journal of Dairy Science 74(2): 409-413. http://dx.doi.org/10.3168/jds.S0022-0302(91)78183-6
Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Antibiotic susceptibility of potentially probiotic Lactobacillus species.Journal of Food Protection 61(12): 1636-1643.
Coconnier MH, Liévin V, Bernet-Camard M F, Hudault S, Servin AL (1997) Antibacterial effect of the adhering humanLactobacillus acidophilus strain LB. Antimicrobial Agents and Chemotherapy 41(5): 1046-1052.
Denev SA (2006) Role of Lactobacilli in gastrointestinal ecosystem. Bulgarian Journal of Agricultural Science 12(1): 63-114.
de Vrese M, Stegelmann A, Richter B, Fenselau S, Laue C, Schrezenmeir J (2001) Probiotics - compensation for lactaseinsufficiency. American Journal of Clinical Nutrition 73(2 Suppl): 421-S429.
Drago L, Gismondo MR, Lombardi A, de Haën C, Gozzini L (1997) Inhibition of in vitro growth of enteropathogens bynew Lactobacillus isolates of human intestinal origin. FEMS Microbiology Letters 153(2): 455-463.http://dx.doi.org/10.1016/S0378-1097(97)00289-9
Duary RK, Singh YR, Batish VK, and Grover S (2011) Assessing the adhesion of putative indigenous probiotic lactobacillito human colonic epithelial cells. Indian Journal of Medical Research 134(5): 664-671.http://dx.doi.org/10.4103/0971-5916.90992
Goldin BR, Gorbach SL (1992) Probiotics for humans. In: Fuller R (ed.), Probiotics, The scientific basis, Chapman andHall, London, 355-376.
Hudault S, Lievin V, Bernet-Camard MF, Servin AL (1997) Antagonistic activity exerted in vitro and in vivo by Lactobacilluscasei (strain GG) against Salmonella typhimurium C5 infection. Applied and Environmental Microbiology 63(2): 513-518.
Hummel AS, Hertel C, Holzapfel WH, Franz C (2007) Antibiotic resistance of starter and probiotic strains of Lactic acidbacteria. Applied and Environmental Microbiology 73(3): 730-739. http://dx.doi.org/10.1128/AEM.02105-06
Isolauri E, Sütas Y, Kankaanpää P, Arvilommi H, Salminen S (2001) Probiotics: effects of immunity. American Journal ofClinical Nutrition 73 (2 Suppl): S444-S450.
Jacobsen CN, Nielsen VR, Hayford AE, Mөller PL, Michaelsen KF, Pærregaard A, Sandström B, Tvede M, Jakobsen M(1999) Screening of probiotic activities of forty-seven strains of Lactobacillus sp. by in-vitro techniques andevaluation of colonization ability of five selected strains in human. Applied and Environmental Microbiology 65:4949-4956.
Liong MT, Shah NP (2005) Acid and Bile Tolerance and Cholesterol Removal Ability of Lactobacilli Strains. Journal ofDairy Science 88(1): 55-66.
Makras L, Triantafyllou V, Fayol-Messaoudi D, Adriany T. Zoumpopoulou G, TsakalidouE, Servin A, De vuyst L (2006)Kinetic analysis of the antibacterial activity of probiotic lactobacilli towards Salmonella enteric serovar typhimuriumreveals a role for lactic acid and other inhibitory compounds. Research in Microbiology 157: 241-247.
McAuliffe O, Cano RJ, Klaenhammer TR (2005) Genetic Analysis of Two Bile Salt Hydrolase Activities in Lactobacillusacidophilus NCFM. Applied and Environmental Microbiology 71(8): 4925-4929.http://dx.doi.org/10.1128/AEM.71.8.4925-4929.2005
Moubareck C, Gavini F, Vaugien L, Butel MJ, Doucet-Populair EF (2005) Antimicrobial susceptibility of Bifidobacterium.Journal of Antimicrobial Chemotherapy 55(1): 38-44. http://dx.doi.org/10.1093/jac/dkh495
Ng SC, Hart AL, Stagg A J, Knight SC (2009) Mechanisms of action of probiotics: recent advances. Inflammatory BowelDiseases 15: 300-310. http://dx.doi.org/10.1002/ibd.20602
Olsen A, Halm M, Jakobsen M (1995) The antimicrobial activity of lactic acid bacteria from fermented maize (kenkey)and their interactions during fermentation. Journal of Applied Bacteriology 79(5):506-512.http://dx.doi.org/10.1111/j.1365-2672.1995.tb03170.x
Pochapin M (2000) The effect of probiotics on Clostridium difficile diarrhoea. American Journal of Gastroenterology95(suppl.): S11-S13.
REFERENCES
8
Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)
Ridwan B, Koning C, Besselink M, Timmerman H, Brouwer E, Verhoef J, Gooszen H, Akkermans L (2008) Antimicrobialactivity of a multispecies probiotic (Ecologic 641) against pathogen isolated from infected pancreatic necrosis.Letters in Applied Microbiology 46(1): 61-67. http://dx.doi.org/10.1111/j.1472-765X.2007.02260.x
Rattanachaikunsopon P, Phumkhachorn P (2010) Lactic acid bacteria: their antimicrobial compounds and their uses infood production. Annals of Biological Research 1(4): 218-228.
Sareml F, Sarem-Damerdjil LO, Nicoias JP (1996) Comparison of the adherence of three Lactobacillus strains to Caco-2and lnt-407 human intestinal cell lines. Letters in Applied Microbiology 22(6): 439-442.http://dx.doi.org/10.1111/j.1472-765X.1996.tb01198.x
Servin AL (2004) Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMSMicrobiology Reviews 28(4): 405-440. http://dx.doi.org/10.1016/j.femsre.2004.01.003
Shah NP (2007) Functional cultures and health benefits. International Dairy Journal 17(11): 1262-1277.http://dx.doi.org/10.1016/j.idairyj.2007.01.014
Sifour M, Tayeb I, Houria O Haddar N, Salima A (2012) Production and characterization of bacteriocin of Lactobacillusplantarum f12 with inhibitory activity against Listeria monocytogenes. The Online Journal of Science and Technology2(1): 55-61.
Šušković J, Kos B, Beganović J, Pavunc AL, Habjanić K, Matošić S (2010) Antimicrobial activity - The most importantproperty of probiotic and starter lactic acid bacteria. Food Technology and Biotechnology 48(3): 296-307.
Temmerman R, Pot B, Huys Y, Swings J (2002) Identification and antibiotic susceptibility of bacterial isolates fromprobiotic products. International Journal of Food Microbiology 81: 1-10. http://dx.doi.org/10.1016/S0168-1605(02)00162-9
Wollowski I, Rechkemmer G, Pool-Zobel BL (2001) Protective role of probiotics and prebiotics in colon cancer.American Journal of Clinical Nutrition 73(2 Suppl): 451S-455S.
Yoshiyuki I, Yasushi K, Kensuke A, Yoshiko H, Takashi S, Tadao S (2009) Conjugative Plasmid from Lactobacillus gasseriLA39 That Carries Genes for Production of and Immunity to the Circular Bacteriocin Gassericin A. Applied andEnvironmental Microbiology 75(19): 6340-6351. http://dx.doi.org/10.1128/AEM.00195-09
Zinedine A, Faid M (2007) Isolation and characterization of strains of Bifidobacteria with probiotic properties in vitro.World Journal of Dairy and Food Sciences 2(1): 28-34.
Lactobacillus acidophilus Suşlarının Potansiyel Probiyotik EtkilerininKarşılaştırmalı Araştırılması
ÖzetGiriş: Probiyotikler, canlı mikrobik gıda takviyeleridir. İddia edilen sağlık destekleyici özellikleri sebebiyle, muazzam birpiyasa değerine ve biyolojik potansiyele sahiptirler. Bu yüzden bu çalışmada, seçilmiş üç Lactobacillus acidophilussuşunun probiyotik potansiyeli karşılaştırmalı olarak çalışılmıştır.Metot: Seçilmiş Lactobacillus acidophilus suşları (NCIM 2660, NCIM 2903 ve NCIM 2285), pH (pH2.5) ve oxgall (%0.3)toleransları, Caco-2 hücrelerine yapışmaları, test mikroorganizmalarına karşı antimikrobiyal özellikleri veantibiyotiklere hassasiyetleri açısından değerlendirildi. Bulgular: NCIM 2903 suşunun öne çıkan özellikleri; yüksek asit toleransı, bütün test mikroorganizmalarına karşıantagonistik aktivite ve Caco-2 hücrelerine yapışması (100 Caco-2 hücresine 225±33 hücrenin yapışması) idi. NCIM2285 suşu safraya tolerans sergiledi ve kullanılan beş antibiyotiğin varlığında çoğalamadı. NCIM 2285 suşu Caco-2hücrelerine orta dercede yapıştı. NCIM 2660 suşu, antibiyotik diskleri olmayan kontrollerle kıyaslandığında, on ikiantibiyotiğin hiçbirinde çoğalamadı. Bu arzu edilen bir durumdu. Ancak, zayıf yapışma özelliği sergiledi. Her üç suş içinoptimum çoğalma sıcaklığı 37ºC olarak tespit edildi.Sonuç: Üç suşun probiyotik özelliklerinde önemli farklılıklar olduğu bulundu. Son değerlendirmede, NCIM 2903suşunun potansiyel bir probiyotik olarak kullanılabileceği düşünülebilir. Bu çalışmalar, daha sonraki uygulamalarda,probiyotik mikroorgamizmaların daha rasyonel olarak seçilmelerinde yardımcı olacaktır.
Anahtar Kelimeler: Antibiyotik hassasiyeti, antimikrobiyal aktivite, asit ve safra toleransı, hücre yapışması, laktik asitbakterileri.
9
Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)